The objective of this research is to study in depth the enzyme adenine phosphoribosyl transferase (APRT), its genetics, and regulation. A large number of mutants, selected as 2,6 diaminopurine resistant, have been isolated from CHO and L-cells. The alterations in enzyme structure will be probed by studying enzyme kinetics, electrophoretic patterns, and finally peptide maps. DNA transformation using mouse and hamster DNA will be used to isolate the APRT gene. Transformed cell DNA will be digested with appropriate restriction enzymes to plasmid pBR322 and amplified in E. coli. Such amplified DNA will then be used in turn to transform APRT minus cells, and the gene isolated from these fragments. Initial experiments are now in progress. Strains of CHO APRT minus have been isolated at a relatively high frequency. There has been much discussion as to whether mutation occurs in two steps, via a heterozyotic stage, or whether CHO has only one functional copy of the gene. These alternatives are being examined by anti-APRT antibody titrations.